divergent control of two type vi secretion systems by rpon

Divergent Control of Two Type VI Secretion Systems byRpoN in Pseudomonas aeruginosaThibault G. Sana, Chantal Soscia, Celine M. Tonglet, Steve Garvis¤, Sophie Bleves*

Laboratoire d’Ingenierie des Systemes Macromoleculaires (UMR7255), CNRS & Aix-Marseille Univ, Marseille, France

Abstract

Three Type VI Secretion System (T6SS) loci called H1- to H3-T6SS coexist in Pseudomonas aeruginosa. H1-T6SS targetsprokaryotic cells whereas H2-T6SS mediates interactions with both eukaryotic and prokaryotic host cells. Little is knownabout the third system, except that it may be connected to H2-T6SS during the host infection. Here we show that H3-T6SS isrequired for P. aeruginosa PAO1 virulence in the worm model. We demonstrate that the two putative H3-T6SS operons,called ‘‘left’’ and ‘‘right’’, are coregulated with H2-T6SS by the Las and Rhl Quorum Sensing systems. Interestingly, the RpoNs54 factor has divergent effects on the three operons. As for many T6SSs, RpoN activates the expression of H3-T6SS left.However, RpoN unexpectedly represses the expression of H3-T6SS right and also H2-T6SS. Sfa2 and Sfa3 are putativeenhancer binding proteins encoded on H2-T6SS and H3-T6SS left. In other T6SSs EBPs can act as s54 activators to promoteT6SS transcription. Strikingly, we found that the RpoN effects of H3-T6SS are Sfa-independent while the RpoN mediatedrepression of H2-T6SS is Sfa2-dependent. This is the first example of RpoN repression of a T6SS being mediated by a T6SS-encoded EBP.

Citation: Sana TG, Soscia C, Tonglet CM, Garvis S, Bleves S (2013) Divergent Control of Two Type VI Secretion Systems by RpoN in Pseudomonas aeruginosa. PLoSONE 8(10): e76030. doi:10.1371/journal.pone.0076030

Editor: Christophe Beloin, Institut Pasteur, France

Received November 12, 2012; Accepted August 24, 2013; Published October 21, 2013

Copyright: � 2013 Sana et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: TGS was financed with a PhD fellowship from the French Research Ministry and with a ‘‘Teaching & Research’’ fellowship (ATER) from AMU. This workwas supported by ‘‘Pathomics’’ ERA-net PATHO (ANR-08-PATH-004-01). The funders had no role in study design, data collection and analysis, decision to publish,or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

¤ Current address: Laboratoire de Biologie Moleculaire de la Cellule (UMR5239), CNRS-ENS Lyon-UCBL-HCL, Lyon, France

Introduction

Protein secretion is an essential for host colonization by

pathogenic bacteria. Multiple systems have evolved in order to

secrete proteins into the extracellular medium or directly into

target cells [1]. The most recently described system, the Type Six

Secretion System (T6SS), was first discovered in Vibrio cholerae [2]

and in Pseudomonas aeruginosa [3]. A unique feature of T6SSs is their

capacity to deliver toxic proteins into eukaryotic host cells as well

as into bacteria [4,5]. These systems were originally thought of as

virulence determinants towards eukaryotic host cells [6,7],

although now they have also been shown to be unambiguously

involved in interbacterial interactions and competition [8].

Nevertheless, the anti-prokaryotic T6SSs may also facilitate the

colonization of specific niches where pathogens can then express

virulence towards eukaryotic cells. Another striking feature of

T6SSs is that multiple distinct T6SS loci are often present in a

single genome. For example, the genomes of Burkholderia

pseudomallei [9,10] and Yersinia pseudotuberculosis [11] harbor six

and four T6SSs respectively. The various T6SS within a single

strain may serve different functions and/or be differentially

regulated. In Burkholderia thailandensis, the T6SS-5 was shown to

be required for virulence in a murine melioidosis model, while

inactivation of T6SS-1 rendered B. thailandensis more susceptible to

contact with other bacteria [12].

The genome of the P. aeruginosa contains three T6SS loci, called

H1- to H3-T6SS [3,7]. H1-T6SS has been widely studied and was

shown to deliver three bacteriolytic toxins to the periplasm of

target bacteria [8,13]. It may give P. aeruginosa a survival benefit in

a multi-bacterial environment. However, less is known about the

two other T6SSs. The H2-T6SS locus of the PAO1 strain of P.

aeruginosa promotes bacterial internalization into epithelial cells,

and indeed plays a role in virulence in the worm model [14]. H2-

T6SS also mediates interbacterial competition through Tle5/PldA

[15], a phospholipase D which was previously shown to contribute

to P. aeruginosa persistence in a chronic pulmonary infection model

[16]. As with many P. aeruginosa virulence factors, the H2-T6SS

machinery is controlled by Quorum-Sensing (QS) and iron

availability in the environment [14]. H2-T6SS and H3-T6SS loci

in the PA14 strain of P. aeruginosa differ from their counterparts in

the PAO1 strain by the absence of three and one putative effector

genes [17]. PA14 H2-T6SS and H3-T6SS are both required for

virulence in the plant model Arabidopsis thaliana. In a mouse model

of acute infection, while a H2-T6SS mutant was affected, a H3-

T6SS mutant was as virulent as the Wild-Type (WT) strain.

Interestingly, the double H2- and H3-T6SS mutant exhibited

dramatically reduced virulence, this suggesting compensation

in vivo between the two systems [17].

In this study, we aimed at determining whether the H2-T6SS

and H3-T6SS loci of PAO1 are related at a transcriptional level.

We observed that, like H2-T6SS, the two H3-T6SS gene clusters

are activated by Quorum Sensing, but are under divergent control

by RpoN.

PLOS ONE | www.plosone.org 1 October 2013 | Volume 8 | Issue 10 | e76030

Table 1. Strains, plasmids and oligonucleotides used in this study.

Strain, plasmid oroligonucleotide

Genotype, descriptionor sequence Source and/or reference

E. coli strains

TG1 supE, D(mcrB-hsdSM)5, thi-1, D(lac-proAB), F9 (traD36, proAB+, lacIq, lacZDM15) Laboratory collection

CC118(lpir) (lpir) D(ara-leu), araD, DlacX74, galE, galK, phoA-20, thi-1, rpsE, rpoB,Arg(Am), recA1, Rfr (lpir)

[42]

TOP10F’ F- mcrA D(mrr-hsdRMS-mcrBC) Q80lacZDM15 DlacX74 nupG recA1 araD139 D(ara-leu)7697galE15 galK16 rpsL(StrR) endA1 l-

Laboratory collection

P. aeruginosa strains

PAO1 Wild-type, prototroph, chl-2 B. Holloway

PAO1Z Promoterless lacZ gene integrated at ctx att site in PAO1 This work

PAO1TS2 H2-T6SS promoter integrated at ctx att site in PAO1 [14]

PAO1TS19 H3-T6SS left promoter integrated at ctx att site in PAO1 This work

PAO1TS20 H3-T6SS right promoter integrated at ctx att site in PAO1 This work

PAO1DclpV2 clpV2 deletion mutant [14]

PAO1DclpV3 clpV3 deletion mutant [20]

PAO1DclpV2DclpV3 clpV2 and clpV3 deletion mutant This work

PAO6358 PAO1 rpoN deletion mutant [28]

PAO6360 PAO1DrpoN att Tn7::rpoN+ GmR [28]

PAO1R lasR mutant of PAO1, CbR [24]

PDO100 rhlI mutant of PAO1, HgR [25]

PAO6358TS2 H2-T6SS promoter integrated at ctx att site in PAO1DrpoN This work

PAO6358TS19 H3-T6SS left promoter integrated at ctx att site in PAO1DrpoN This work

PAO6358TS20 H3-T6SS right promoter integrated at ctx att site in PAO1DrpoN This work

PAO6360TS2 H2-T6SS promoter integrated at ctx att site in PAO1DrpoN att Tn7::rpoN+ This work

PAO6360TS19 H3-T6SS left promoter integrated at ctx att site in PAO1DrpoN att Tn7::rpoN+ This work

PAO6360TS20 H3-T6SS right promoter integrated at ctx att site in PAO1DrpoN att Tn7::rpoN+ This work

PAORTS19 H3-T6SS left promoter integrated at ctx att site in PAOR This work

PAORTS20 H3-T6SS right promoter integrated at ctx att site in PAOR This work

PDO100TS19 H3-T6SS left promoter integrated at ctx att site in PDO100 This work

PDO100TS20 H3-T6SS right promoter integrated at ctx att site in PDO100 This work

PAO1sfa2 sfa2 mutant of PAO1, CbR This work

PAO sfa2TS2 H2-T6SS promoter integrated at ctx att site in PAO1sfa2 This work

PAO sfa2TS19 H3-T6SS left promoter integrated at ctx att site in PAO1sfa2 This work

PAO sfa2TS20 H3-T6SS right promoter integrated at ctx att site in PAO1sfa2 This work

PAO1sfa3 sfa3 mutant of PAO1, CbR This work

PAO sfa3TS2 H2-T6SS promoter integrated at ctx att site in PAO1sfa3 This work

PAO sfa3TS19 H3-T6SS left promoter integrated at ctx att site in PAO1sfa3 This work

PAO sfa3TS20 H3-T6SS right promoter integrated at ctx att site in PAO1sfa3S This work

Plasmids

pCR2.1 TA cloning, lacZa, ColE1, f1 ori, ApR KmR Invitrogen

pMini-CTX::lacZ V-FRT-attP-MCS, ori, int, oriT, TcR [18]

pMP220 Broad host-range lacZ transcriptional fusion, TcR Laboratory collection

pRK2013 Tra+, Mob+, ColE1, KmR Laboratory collection

pKNG101 oriR6K, mobRK2, sacBR+, SmR (suicide vector) [43]

pJN105 GmR, araC-pBAD [44]

pTS2 722 bp upstream region of H2-T6SS in pMini-CTX::lacZ [14,25]

pTS12 486 bp upstream region of H3-T6SS left in pCR2.1 This work

pTS13 494 bp upstream region of H2-T6SS right in pCR2.1 This work

pTS19 486 bp upstream region of H3-T6SS left in pMini-CTX::lacZ This work

pTS20 494 bp upstream region of H2-T6SS right in pMini-CTX::lacZ This work

RpoN Control of P. aeruginosa T6SSs


Materials and Methods

Bacterial Strains, Plasmids, and Growth ConditionsThe bacterial strains and plasmids used in this study are

described in Table 1. LB and TSB broths and agar were used for

the growth of P. aeruginosa and Escherichia coli strains at 37uC.

Cultures were inoculated at an optical density at 600 nm (OD600)

of 0.1 with overnight cultures, and strains were grown at 30, 37 or

42uC with aeration in TSB. Recombinant plasmids were

introduced into P. aeruginosa using the conjugative properties of

pRK2013 (Table 1) or by electroporation. Pseudomonas transconju-

gants were selected on Pseudomonas isolation agar (PIA, Difco

Laboratories) supplemented with appropriate antibiotics. The

antibiotic concentrations were as follows: for E. coli, ampicillin

(50 mg ml–1), kanamycin (25 mg ml–1), tetracycline (15 mg ml–1),

gentamicin (10 mg ml–1); for P. aeruginosa, tetracycline (200 mg ml–1

for plates or 50 mg ml–1 for liquid growth), gentamicin (50 mg ml–

1), carbenicillin (500 mg ml–1).

lacZ Reporter Fusion and b-galactosidase AssayThe H3-T6SS left-lacZ and H3-T6SS right-lacZ transcriptional

fusions were constructed by PCR amplification of respectively 486

and 494 bp upstream DNA region from the lip3 or hsiB3 gene by

using TSO15/TSO16 and TSO17/TSO18 primers (Table 1).

PCR amplification products were directly cloned into the pMini-

CTX::lacZ vector [18], yielding pTS12 and pTS13, in pCR2.1,

and pTS19 and pTS20 in MiniCTX-‘lacZ. Nucleotide sequences

were verified by sequencing (GATC). The promoter fragment was

integrated at the CTX phage attachment site in PAO1 and

isogenic mutants using established protocols [18].

Overnight culture, grown in TSB, was diluted in TSB to

OD600 = 0.1. Growth and ß-galactosidase activity were monitored

by harvesting samples at different time intervals. ß-galactosidase

activity was measured according Miller [19], based on o-

nitrophenyl-b-D-galactopyranoside hydrolysis. ß-galactosidase ac-

tivities were expressed in Miller units.

Construction of the DclpV2DcplV3 MutantTo generate the DclpV2cplV3 mutant, the pTS27 mutator

plasmid [14] was mobilized in the P. aeruginosa strain PAO1DclpV3

[20]. Mutants which had undergone a double recombination

event, resulting in the non-polar deletion of the clpV2 gene, were

verified by PCR with the primers OA14 and OA17 that flank

clpV2.

Construction of sfa2 and sfa3 MutantsTo generate sfa2 and sfa3 mutants, internal fragments of 490

and 511 bp were respectively amplified with TSO118-TSO119

and TSO120-TSO121 and cloned in the pCR2.1, resulting in

pSBC56 and pSBC57. The mutator plasmids were electroporated

into P. aeruginosa PAO1 and the mutant bacteria selected on PIA

medium containing Carbenicillin. The insertions were verified

by PCR with the primer pairs TSO39-TSO40-TSO45 and

Table 1. Cont.

Strain, plasmid oroligonucleotide

Genotype, descriptionor sequence Source and/or reference

pTS25 sfa2 gene in pCR2.1 This work

pTS27 500 bp upstream and 500 bp downstream clpV2 in pKNG101 [14]

pSBC52 sfa2 gene in pJN105 This work

pSBC56 490 bp internal fragment of sfa2 cloned in pCR2.1 This work

pSBC57 511 bp internal fragment of sfa3 cloned in pCR2.1 This work

pMAL.R PlasR-lacZ transcriptional fusion in pMP220 [14,24]

pMAL.V PrhlR-lacZ transcriptional fusion in pMP220 [24]

Oligonucleotides

TSO15 59-CCAGGCTCCATACCGCGAACTG-39 This work

TSO16 59-GGCGGCTGACTCCGATGCAA-39 This work

TSO17 59-TTGCTGTCGTCGCCGCTGAT-39 This work

TSO18 59-GGGAGTCCAACGAAAATTTTATTTTGC-39 This work

TSO39 59-ATGTCCGTCATCACCCATCCCCACG-39 This work

TSO40 59-TCAGGTCCGGGGATCGCCGAAATG-39 This work

TSO41 59-ATGTTCAGCCGCGTACCGCAACC-39 This work

TSO42 59-TCACTTGCCCACCAGCGAGACCACG-39 This work

TSO45 59-CATGCGACGCTGGGCGAGCACG-39 This work

TSO46 59-AATCTATGGGTTCCTGGGGCAAGATGGG-39 This work

TSO118 59-ACCTGCAGGGAT TC CCCATCC-39 This work

TSO119 59-TGCAACACG CGCAACAGCTTGG-39 This work

TSO120 59-TCGTCGCGGTCAACTGCGGTGC-39 This work

TSO121 59-TTCTCCAGCTCG CGGATATTGC-39 This work

OA14 59-GGAAAGCTTTTCGCCCTCGTCGGATTG-39 [14]

OA17 59-AAAGAATTCGAGGCGTTGCAGCAGATG-39 [14]

doi:10.1371/journal.pone.0076030.t001



TSO41-TSO42-TSO46 that hybridize outside and inside of the

sfa2 and sfa3 genes.

Caenorhabditis elegans Killing AssayThe slow killing assay was performed as described previously

[14]. Each independent assay consisted of three replicates. E. coli

OP50 was used as a control. L4 to adult stage C. elegans were

removed from food and placed on unseeded NGM plates for 24

hours at 25uC. 50 worms were then picked onto plates containing

overnight grown bacteria. Worms were evaluated for viability on a

daily basis. Animal survival was plotted using the PRISM 5.0

program. Survival curves are considered significantly different

from the control when P-values are ,0.05. Prism calculates

survival fractions using the product limit (Kaplan-Meier) method.

Prism compares survival curves by two methods: the log-rank test

(also called the Mantel-Cox test) and the Gehan-Breslow-

Wilcoxon test.

Statistical AnalysisPaired Student’s t tests were performed using Excel software. In

the figures, * means P-values #0.05, ** #0.01, and *** #0.001.

Results

H2 and H3-T6SS are Both Involved in Virulence TowardsC. elegans

Functional compensation between PA14 H2- and H3-T6SS has

been observed in a lung infection model [17]. We previously

showed that the H2-T6SS of PAO1 mediates virulence in a worm

model [14]. Therefore, we wondered whether the H3-T6SS of

PAO1 is also required for virulence. We indeed found decreased

virulence in a PAO1 H3-T6SS mutant, which harbors a deletion

of the clpV3 gene (Fig. 1). clpV3 encodes the ClpV AAA+ ATPase

[20], a core component of the secretion machinery (Fig. 1). The

worms infected with the mutant appeared to die with a 2 day delay

when compared to the WT strain. H3-T6SS is thus necessary for

killing C. elegans, however to a lesser extent than H2-T6SS [14]. In

contrast with the findings for PA14 [17], the virulence of the

double mutant DclpV2DclpV3 was not lower than in the single

mutants. Both T6SS are thus required for virulence in PAO1.

Figure 1. H3-T6SS is required for virulence in C. elegans. C.elegans was infected with PAO1 and isogenic DclpV2, DclpV3 orDclpV2DclpV3 mutants. The resulting C. elegans survival curve is shownfor each strain. Evalue ,0.01 (Prism 5 software).doi:10.1371/journal.pone.0076030.g001

Figure 2. The P. aeruginosa PAO1 H2- and H3-T6SS gene clusters. A. H2-T6SS is organized in one putative operon (from [14]). B. H3-T6SS isorganized in two putative operons. The genes are labeled hsiA3 to hsiJ3 for the left operon and hsiB3 to hsiH3 for the right operon and, whereapplicable, with the given name, i.e., clpV3 or sfa3. Gene annotation numbers are also indicated (e.g. PA2359). The promoter region of each operon isalso shown. C. The intergenic sequence between lip3 and hsiB3 genes is represented. The –35 box and the –10 box of the s70 promoters predicted byBprom are highlighted in green and red respectively. The translation initiation codons of lip3 and hsiB3 genes are underlined. 486 bp of the leftoperon upstream region and 494 bp of the right operon upstream region were used for the transcriptional fusions, which are encoded by pTS19 andpTS20 respectively.doi:10.1371/journal.pone.0076030.g002



H2-T6SS and H3-T6SS are Coregulated by Quorum-Sensing

While the H1-T6SS is not expressed in a PAO1 WT

background [3,21], H2-T6SS expression is controlled by QS in a

cell-density dependent manner [14] (Fig. 2A). We therefore

investigated the transcriptional regulation of the H3-T6SS gene

cluster in PAO1. The H3-T6SS locus is organized into two

divergent gene clusters (Fig. 2B), and we analyzed the intergenic

DNA region between lip3 (PA2364) and hsiB3 (PA2365) for

potential regulatory elements (Fig. 2C). The BProm algorithm

identified one s70 dependent promoter upstream of the lip3 gene

and another, in the opposite direction, upstream of the hsiB3 gene

(http://linux1.softberry.com/berry.phtml?topic = bprom&group =

programs&subgroup = gfindb) (Fig. 2C). To construct chromosomal

Figure 3. H2-T6SS and H3-T6SS are coregulated by QS. (A) Expression patterns of the H2-T6SS, H3-T6SS left, and H3-T6SS right lacZtranscriptional fusions from the WT PAO1 strain (PAO1TS2, PAO1TS19 and PAO1TS20 respectively) and of a control strain (PAO1Z). Expression is givenin Miller Units at different time points during growth at 37uC in TSB medium (see Material and Methods). The growth phases are represented hereand the growth curves are presented in Fig. S1. Expression of H3-T6SS left (B) and of H3-T6SS right (C) is shown in the WT (blue bars) or in QS mutantstrains after 4 hours of growth at 37uC. The PAOR background (red bars) is a lasR mutant and PDO100 (green bars) a rhlI mutant. Each experiment wasdone in triplicate and independently repeated three times; error bars indicate the standard deviation.doi:10.1371/journal.pone.0076030.g003



transcriptional lacZ fusions, the regions upstream of the ATG of lip3

and hsiB3, the first gene of each of the two H3-T6SS clusters

respectively (hereinafter called ‘‘left’’ and ‘‘right operons’’ for

simplicity) were fused to lacZ and then integrated at the CTX phage

attB site as a single copy on the chromosome, yielding strains

PAO1TS19 and PAO1TS20 respectively (Table 1). Strain PAO1Z

was similarly constructed by integrating a promoter-less lacZ gene to

serve as a negative control. The b-galactosidase activity profiles

associated with the two H3-T6SS transcriptional fusions were

similar. Expression was induced at the transition from log-to-

stationary phase and a maximal level was reached late in stationary

phase, after 9 hours of growth (Fig. 3A and Fig. S1). The expression

then stayed and maximal for at least 4 more hours. No promoter

activity was detectable in the control strain PAO1Z. Moreover, H3-

T6SS was clearly more expressed at 37uC than at the other tested

temperatures, 30uC and 42uC (Fig. S1). As previously observed [14],

H2-T6SS expression was also induced at the transition phase, but a

maximal and steady level of expression was reached earlier in

stationary phase (Fig. 3A and Fig. S1).

The cell density dependent expression profile of the H3-T6SS

reporter fusions suggested that the transcription of the H3-T6SS

might be regulated by QS. This would be in line with the LasR –

mediated regulation of hcp3 in the H3-T6SS right operon of PA14

[17], and with two transcriptomic studies indicating de-regulated

expression of genes in the right H3-T6SS operon of PAO1 in QS

mutants [22,23]. We therefore examined the expression of the two

H3-T6SS fusions in P. aeruginosa QS mutants, a lasR mutant

(PAOR) [24] and a rhlI mutant (PDO100) [25]. Compared to the

WT PAO1 strain (Fig. 3B & 3C), the expression of the left and

right H3-T6SS operons was significantly decreased in the lasR

mutant (2.7-fold and 3.3-fold respectively) and in the rhlI mutant

(2.4-fold and 2.7-fold respectively). In conclusion, H3-T6SS is

coregulated with H2-T6SS by the Las and Rhl QS systems.

Divergent Effects of RpoN on H2- and H3-T6SS GeneExpression

Several reports in the literature have shown that T6SS

transcriptional activity requires the sigma factor RpoN (s54)

and cognate activators encoded within the T6SS operon. In the

initial genetic screen that led to the discovery of T6SS genes in V.

cholerae [2], one of the attenuated mutants in virulence towards

Dictyostelium was in the vasH gene which encodes a s54-activator.

Similarly, in Aeromonas hydrophila, a VasH homologue was shown to

be required for cytotoxicity towards macrophages and epithelial

cells since it was found to be essential for the expression of the

genes encoding the T6SS machinery [26]. In agreement, Bernard

and coworkers demonstrated, in a reconstituted heterologous

system, that s54-activators from various T6SSs together with the

E. coli s54-RNAP (RNA polymerase) complex allowed expression

of T6SS genes [27].

We thus monitored expression of the H2-T6SS and H3-T6SS

lacZ fusions in the rpoN mutant (Table 1) at the entry to stationary

phase (Fig. 4). The rpoN mutation is complemented (PAO6360) or

not (PAO6358) on the chromosome [28]. Expression of the left

Figure 4. Divergent control of RpoN on H2-T6SS and H3-T6SS.The expression of H2-T6SS (A), H3-T6SS left (B), and H3-T6SS right (C)after 7 hours (A), or 9 hours (B & C) of growth at 37uC in the WT strain(blue bars), in a PAO1DrpoN mutant complemented strain (green bars,PAO6360 strain) or the PAO1 DrpoN mutant (red bars, PAO6358 strain).Expression is given in Miller units. Each experiment was done intriplicate and independently repeated three times; error bars indicatethe standard deviation.doi:10.1371/journal.pone.0076030.g004



H3-T6SS operon decreased 2.0-fold in the rpoN mutant, and

complementation of the mutation restored WT expression levels

(Fig. 4B). This means that expression of the left H3-T6SS operon

depends on RpoN, and may require a s54-activator as observed

in for other T6SSs. Unexpectedly, H2-T6SS and the right H3-

T6SS operons were overexpressed in the rpoN mutant (4.5-fold and

4.7-fold increase respectively). Expression was restored to WT

levels upon complementation (Fig. 4A & 4C). This suggests that

RpoN mediates the transcriptional repression of these two

operons. In conclusion, RpoN has divergent roles in regulating

P. aeruginosa T6SS gene expression.

Sfa2 Decreases H2-T6SS Expression whereas H3-T6SSExpression is Sfa-independent

In P. aeruginosa, the sfa2 and sfa3 genes (sigma factor activator)

from the H2- and H3-T6SS loci (Fig. 2A & 2B) encode putative

RpoN activators also called EBPs (enhancer binding protein) [7].

As for other EBPs, Sfa2 and Sfa3 contain two Walker A and B

motifs that have roles in nucleotide binding and hydrolysis, and

the highly conserved ‘‘GAFTGA’’ domain that is indispensable for

the nucleotide-dependent interactions with s54-RNAP that drives

open complex formation and transcription (Fig. S2) [29,30]. We

thus wondered if Sfa2 and Sfa3 proteins could be involved in H2-

and H3-T6SS regulation. To test this hypothesis, sfa2 and sfa3

mutants were constructed and expression of the three lacZ fusions

were assayed in the mutant backgrounds. While mutations in sfa2

and sfa3 had no effect on H3-T6SS expression levels (Fig. 5A &

5B), H2-T6SS expression was increased in the sfa2 mutant (3.7-

fold). Expression could be restored to WT levels upon comple-

mentation in trans with a WT copy of sfa2 (Fig. 5C). Moreover Sfa3

has no effect on H2-T6SS expression (Fig. 5C). Taken together,

these data suggest that H3-T6SS expression is Sfa-independent and

Figure 5. Sfa2 negatively controls H2-T6SS expression while H3-T6SS expression is Sfa-independent. The expression of H3-T6SS left (A),H3-T6SS right (B), and H2-T6SS (C) after 6 hours of growth after 9 hours (A & B) or 7 hours (C) at 37uC in the WT strain (blue bars), in a PAO1sfa2mutant (red bars), and in a PAO1sfa3 mutant (green bars). Expression is given in Miller units. Each experiment was done in triplicate andindependently repeated three times; error bars indicate the standard deviation.doi:10.1371/journal.pone.0076030.g005



that the H2-T6SS operon may be indirectly repressed by RpoN in

a Sfa2-dependent manner.

Sfa2 Contributes to Repression of H2-T6SS by RpoNTo demonstrate that the RpoN repression of H2-T6SS is

mediated by Sfa2, we hypothesized that overproduction of Sfa2

should have no effect in a rpoN mutant while it should decrease

expression in a WT background. We thus monitored H2-T6SS

expression upon Sfa2 overproduction in exponential phase in these

two backgrounds. We chose to probe expression at this particular

moment of growth to focus on the effect of the overproduced Sfa2

and not of the chromosomal sfa2 gene, which not yet fully

expressed at this time. In support of our hypothesis we indeed

found that Sfa2 overproduction led to the decreased expression of

H2-T6SS in the WT, and had no effect in the rpoN background

(Fig. 6). Hence Sfa2 mediates to the indirect repression of H2-

T6SS by RpoN.

Discussion

The P. aeruginosa genome harbors three T6SS clusters. The H1-

T6SS of the PAO1 strain targets toxins into host bacteria [8,13]

and H2-T6SS is involved in interactions with eukaryotic and

prokaryotic hosts [14,15]. The role of H3-T6SS has been mainly

studied in the PA14 strain of P. aeruginosa, which appears to differ

from PAO1 at the H2- and H3-T6SS gene level. The PA14 H3-

T6SS is required for virulence in the plant model A. thaliana and

may compensate for the loss of H2-T6SS in mouse virulence

because only the double H2-T6SS H3-T6SS mutant is less

virulent than the WT strain [17]. Here, we have shown that H3-

T6SS is required for P. aeruginosa PAO1 virulence towards worms.

However, we did not observe any compensation between the two

T6SSs, at least in the worm model. But the clpV3 mutation

phenotype is dominant on clpV2 since the double mutant has the

same virulence defect as the clpV3 mutant this suggesting a genetic

interaction. Moreover, we found that RpoN divergently regulates

expression of PAO1 T6SSs, by repressing H2-T6SS and the right

H3-T6SS and activating the left H3-T6SS. Interestingly solely Sfa2,

the s54 activator encoded within the H2-T6SS cluster, partici-

pates in RpoN-control. This is the first example of a RpoN-

repression mechanism mediated by a T6SS-encoded activator.

We observed that like H2-T6SS [14], the two H3-T6SS operons

of the PAO1 strain are induced at the growth phase transition by

LasR and RhlR QS regulators (Fig. 3). This is in line with data

establishing a differential regulation of the three PA14 T6SS loci

by LasR [17,31]. We also noticed that the two H3-T6SS operons

are fully expressed late in stationary phase (Fig. 3A), suggesting

induction by the RpoS sigma factor. In agreement, a transcrip-

tomic study showed that the right H3-T6SS operon is strongly

dependent on RpoS [32], however the authors did not identify the

heptameric consensus sequence CTATACT defined as the - 10 of

RpoS-controlled promoters [33] upstream of the right H3-T6SS

operon. Interestingly, the right H3-T6SS operon also belongs to a

group of genes that are induced by contact with eukaryotic cells

[34]. This is in agreement with our data on the involvement of H3-

T6SS in P. aeruginosa virulence in a eukaryotic model (Fig. 1).

Furthermore, we also observed that H3-T6SS is more highly

expressed at 37uC, the temperature of certain eukaryotic hosts

(Fig. S1). Finally, the H3-T6SS from PAO1 was recently shown to

be dispensable for bacterial competition (Russell 2013). All

together, and in contrast to H2-T6SS, the H3-T6SS machinery

in PAO1 appears to be exclusively dedicated to interactions with

eukaryotic cells.

We also showed a complex RpoN-control of T6SS expression.

Indeed H2-T6SS and the right H3-T6SS operons are unexpect-

edly under a negative control by RpoN, to the contrary of other

T6SSs (for a review see [5]), (Fig. 4A & 4C). Moreover, the RpoN-

mediated repression of H2-T6SS requires Sfa2, the EBP encoded

within H2-T6SS (Fig. 5C & 6). H3-T6SS right is Sfa-independent,

although H3-T6SS left encodes an EBP, Sfa3 (Fig. 5B). We also

showed that the H3-T6SS left operon is activated by RpoN, and

independently of any Sfa (Fig. 5A). Hence the two H3-T6SS

operons are divergently regulated by RpoN. This could fit with the

recent observations of Dong and Mekalanos [35]. In V. cholerae,

they observed that RpoN positively regulates the expression of the

hcp operons and vgrG3 that encode Type VI secreted proteins, but

has no effect on the expression of the main T6SS cluster encoding

sheath and other structural components of the phage tail-like

machinery [5,36]. This is presumably because the latter are

recycled. The role of Sfa3 is also intriguing. It may be required for

coregulation of substrate genes that are not part of the H3-T6SS

locus.

Figure 6. Sfa2 mediates down-regulation of H2-T6SS by RpoNExpression of H2-T6SS after 3.5 hours of growth at 37uC in WT (blue andred bars) or rpoN mutant (purple and light blue bars) strainsoverproducing Sfa2 in trans (red or light blue bars), or without Sfa2overproduction (empty vector, blue or purple bars). Expression is givenin Miller units. For Sfa2 overproduction gene expression from the PBAD

promoter was induced with arabinose (0.5%). Each experiment wasdone in triplicate and independently repeated three times; error barsindicate the standard deviation.doi:10.1371/journal.pone.0076030.g006



In P. aeruginosa, RpoN plays important roles in mobility, in the

transport of nutrients, in the formation of pili, in mucoidy and in

cell-to-cell signaling (for a review see [37]). As a sigma factor,

RpoN positively regulates its target genes (e.g. type IV pili and

flagellum genes), but has also been shown to negatively regulate

QS [28], the expression of sadB, coding an important protein

during biofilm formation [38], and of aceA, coding an isocitrate

lyase, an enzyme required for the metabolic pathway utilized by P.

aeruginosa during chronic pulmonary infections [39]. Unlike RpoN-

mediated activation, RpoN repression is indirect. In agreement we

have been unable to identify RpoN consensus elements in the

promoter regions of H2- and of the right H3-T6SS operons with

Virtual footprint (http://www.prodoric.de/vfp/vfp_promoter.

php), while a RpoN binding site was previously proposed for the

H3 left operon that we found RpoN-activated [27] (Bernard

2011).

Taken together, our data allow us to propose a model in which

the expression of H2-T6SS and H3-T6SS is induced by QS [14] at

the transition between the exponential and stationary phase, with

H3-T6SS reaching full expression later than H2-T6SS in the

stationary phase. The H2-T6SS operon codes Sfa2, that once

produced activates RpoN to repress its own expression. A first

explanation in line with Heulier et al. [28] could have been that

Sfa2 is the EBP which activates RpoN in order to repress QS, thus

arresting H2-T6SS induction. However, we found that lasR and

rhlR transcriptional lacZ fusions were not impacted upon Sfa2

overproduction (Fig. S3). We would thus like to propose that

RpoN together with Sfa2 activates an unknown repressor of H2-

T6SS. Repression of the H3-T6SS right operon might be

explained by the RpoN/GacA/RsmA pathway [40]. Indeed, the

H3-T6SS right operon is strongly activated by GacA and RsmYZ

[41], while RpoN has a negative effect on gacA expression [28] and

thus on expression of H3-T6SS right. Future studies will be

required to decipher this network of regulation.

Supporting Information

Figure S1 H2-T6SS and H3-T6SS are differentiallythermoregulated. The expression pattern of the H2-T6SS-lacZ

(A), H3-T6SS left-lacZ (B), and H3-T6SS right-lacZ (C) transcrip-

tional fusions in the WT PAO1 strain is given in Miller Units at

different time points over the growth and at 3 different

temperatures: 30uC (blue), 37uC (green) and 42uC (red). The

OD600 is also presented (diamonds). A control strain (PAO1Z)

(grey squares) is included for each graph. Each experiment was

done in triplicate and independently repeated three times; error

bars indicate the standard deviation.

(TIF)

Figure S2. Sfa2 and Sfa3 are EBPs. Sfa2 (A) and Sfa3 (B)are 503 and 361 amino acids long. Both proteins possess

WalkerA, Switch ASN, GAFTGA, WalkerB and Arg Fingers

motifs that are specific to s54 activators.

(TIF)

Figure S3 Sfa3 has no effect on QS gene expression.Expression of rhlR-lacZ (A) and lasR-lacZ (B) transcriptional fusions

is given in Miller Units after 4 h of growth in the PAO1 strain

overproducing Sfa2 (red bars) or not (blue bars, empty vector).

Each experiment was done in triplicate and independently

repeated three times; error bars indicate the standard deviation.

(TIF)

Acknowledgments

We thank Dieter Hass for sharing PAO6358 and PAO6360 strains, Rome

Voulhoux, Berengere Ize and Genevieve Ball for constant support, Amel

Latifi and Ben Field for careful correction of the manuscript and TGS

thesis committee for fruitful discussions.

Author Contributions

Conceived and designed the experiments: TGS SB. Performed the

experiments: TGS CS CMT SG. Analyzed the data: SB TGS. Wrote

the paper: SB TGS.

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divergent control of two type vi secretion systems by rpon

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